Pretargeting with Cucurbituril-Adamantane Host-Guest Pair in Xenograft Models.

The goal of reducing the total-body radiation dose of macromolecule-based nuclear medicine with a 2-step pretargeting strategy has been achieved with several pretargeting methodologies in preclinical and clinical settings. However, the lack of modularity, biocompatibility, and in vivo stability in existing pretargeting agents obstructs their respective platforms' wide clinical use. We hypothesized that host-guest chemistry would provide an optimal pretargeting methodology. A cucurbit[7]uril host and an adamantane guest molecule form a high-affinity host-guest complex (association constant, ∼1014 M-1), and in this work, we explored the use of this noncovalent interaction as the basis for antibody-based pretargeted PET. Along with the straightforward modularity of these agents, cucurbit[7]uril and adamantane are recognized to have high in vivo stability and suitability for human use, which is why we proposed this methodology as the ideal approach for pretargeted nuclear medicine. Methods: Three 64Cu-labeled adamantane guest radioligands were developed, and their in vitro stability, lipophilicity, and in vivo blood half-lives were compared. The adamantane radioligands were analyzed for pretargeting using a cucurbit[7]uril-modified carcinoembryonic antigen-targeting full-length antibody, hT84.66-M5A, as the macromolecule pretargeting agent with 2 different dosing schedules. These molecules were evaluated for pretargeting in human pancreatic cancer BxPC3 and MIAPaCa-2 mouse xenografts using PET and in vivo biodistribution studies. The dosimetry of the cucurbit[7]uril-adamantane (CB7-Adma) pretargeting approach in men was calculated and compared with that of the directly 89Zr-labeled hT84.66-M5A. Results: The adamantane radioligands possessed high in vitro stability up to 24 h (>90%). Pretargeted PET with CB7-Adma methodology resulted in specific tumor uptake (P < 0.05) with low background signal. The in vivo formed CB7-Adma complex was demonstrated to be stable, with high tumor uptake up to 24 h after radioligand injection (12.0 ± 0.9 percentage injected dose/g). The total-body radiation dose of the pretargeting strategy was only 3.3% that of the directly 89Zr-labeled hT84.66-M5A. Conclusion: The CB7-Adma strategy is highly suitable for pretargeted PET. The exceptional stability of the pretargeting agents and the specific and high tumor uptake of the pretargeted adamantane radioligands provide great potential for the platform.

Investigation of Copper-64-Based Host-Guest Chemistry Pretargeted Positron Emission Tomography.

Pretargeting is a technique that uses macromolecules as targeting agents for nuclear imaging and therapy with the goal of reducing the radiation toxicity to healthy tissues often associated with directly radiolabeled macromolecules. In pretargeting, a macromolecule is radiolabeled in vivo at the target site using a radiolabeled small molecule (radioligand) that interacts with the macromolecule with high specificity. We report an investigation of host-guest chemistry-driven pretargeting using copper-64 radiolabeled ferrocene (Fc; guest) compounds and a cucurbit[7]uril (CB7; host) molecule functionalized carcinoembryonic antigen targeting hT84.66-M5A monoclonal antibody (CB7-M5A). Two novel ferrocene-based radioligands ([64Cu]Cu-NOTA-PEG3-Fc and [64Cu]Cu-NOTA-PEG7-Fc) were prepared, and their in vitro stability, pharmacokinetic in vivo profile in healthy mice, and pretargeting performance in a subcutaneous BxPC3 human pancreatic cancer cell xenograft mouse model were compared. The antibody dosing was optimized using a zirconium-89 radiolabeled M5A antibody ([89Zr]Zr-DFO-M5A) in a BxPC3 xenograft model, and the dosimetry of [89Zr]Zr-DFO-M5A and the pretargeting approach were compared. Finally, the effects of varying lag times up to 9 days between CB7-M5A and radioligand injection were investigated. In vivo pretargeting studies with both ferrocene radioligands resulted in specific tumor uptake (p = 0.0006 and p = 0.003) and also showed that the host-guest-based pretargeting approach excels with extended lag times up to 9 days with good tumor localization, suggesting that host-guest pretargeting may be suitable for use without clearing agents which have complicated clinical application of this technique. To our knowledge, the reported lag time of 9 days is the longest investigated lag time in any reported pretargeting studies.